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Background

In Malayer city (West Iran), grape (Vitis vinifera L.) is the most significant farming crop and the space under grape cultivation is more than 10000 ha. Among all the fruit crops, the grape has emerged as the most successful commercial crop in the recent years (1). Vine cicada, Psalmocharias alhageos (Hem., Cicadidae), is one of the most important pests of vineyards in Malayer. Main harm of P. alhageos is caused by long feeding time of nymphs on the vine roots and laying eggs of females under the skin of the shoots (2,3).
Imidacloprid 1-(6-chloro-3-pyridylmethyl)-N-nitroimidazoli- din-2-ylideneamine (fig.1), is a systemic neonicotinoid insecticide with soil, seed and foliar uses for the mastery of sucking pests, because the active ingredient is stored in plant tissues for 2 or 3 months (4–6)(7). It is widely used in Malayer vineyards for the control of Psalmocharias alhageos (2)

Figure 1) Structure of imidacloprid
Their chemical properties, particularly their high water solubility and partitioning properties (low log KOW) and low soil adsorption (log KOC), promote movement of these insecticides through the surface and subsurface runoff and result in extended persistence under simulated environmental conditions (8).
It is commonly used on rice, cereals, maize, potatoes, vegetable, sugar beet, fruit, cotton, hops and turfs and is very general once used as a seed or soil treatment. Imidacloprid residues have reportable in numerous agriculture crops (9–12). However, when the appliance of pesticides in agricultural fields, residues will be absorbed and continue plants, inflicting a possible hazard for human health (5). To minimize the adverse result of pesticides residues on human and setting, the food commodities treated with pesticides are strictly controlled by the authorities worldwide.
Imidacloprid is intended to be effective by contact or intake. Imidacloprid acts on many forms of post-synaptic nicotinic neurotransmitter receptors within the system. In insects, these receptors are placed only within the central nervous system. Following unreturn binding to the receptors, nerve impulses are spontaneously discharged initially, followed by failure of the neuron to propagate any signal. Sustained activation of the receptor results from the inability of acetyl cholinesterases to break down the chemical (7,13). Imidacloprid is extremely low in toxicity via dermal exposure, and moderately poisonous if eaten; but upon inhalation, its toxicity is variable (14,15). Toxicological studies of imidacloprid are limited and acceptable daily intake (ADI) was before reported as 0.06 mg/kg/day (14).
Due to the high consumption of imidacloprid in agriculture, now a day, there has been a growing interest in detective work and quantifying of insecticide residues in
agricultural produce supposed for human consumption (16). The increasing society concern over the potential health risk associated with exposure to pesticides has led to the strict regulation of maximum residue limits (MRLs) of pesticide residues in food crops. When these pesticides are applied according to good agricultural practices, MRLs are not exceeded, but their wrong application may leave harmful residues, which involve possible health risk and environmental pollution (17,18).
Aims of the study:
Based on the high use of imidacloprid in the infected vineyards to cicada in Malayer, therefore, it is necessary to ensure that the residue concentration of imidacloprid in grape does not pose any risk to consumers.
The present studies were undertaken to determine the residues of imidacloprid on grape following its applications at the minimum effective and double the minimum effective dose. Data were obtained to cover a range of pre-harvest intervals (PHIs) 21 days and the results were compared with the Codex Maximum Residues Limits (MRLs) proved for this insecticide in grape. The purpose of this study was to imidacloprid residue on grapes, to determine its residue deposit, the half-life of degradation, safe pre-harvest interval and harvest time residues for use of this fruit after its multiple spraying.
 

Materials & Methods

Preparation of the Suspension of imidacloprid and their Application in vineyards
Field experiments were conducted during 2017 on vineyards cultivar at Malayer, Iran. At first, the vineyards infected with Vine cicada were chosen in late July. At this stage, the nymphs of cicada existed from the soil, so Imidacloprid poisoning was sprayed onto the vine trees. Three replicate have been selected for each treatment i.e. control, recommended dose and double the recommended dose. The treatments were untreated control, effective and double effective dose of Confidor 200 SL at 80 and 160 g a.i. ha^-1. For every treatment 10 plants were choose. The spray extent taken was 1000 L ha^-1. Grape control samples have been sprayed with water.
Chemicals and Reagents
Pesticide analytical standard of imidacloprid (purity P≥99.9%) was purchased with the purity certified by using Dr Ehrenstorfer Inc. (Augsburg, Germany). Individual pesticide stock solution (1000 µg ml^-1) have been prepared in pure acetonitrile (MeCN) and stored at −14 ^◦C. Intermediate and working standard solutions of imidacloprid were prepared in acetonitrile. Calibration solutions were prepared with different concentrations simply earlier than the use. HPLC grade acetonitrile and Analytical grade anhydrous magnesium sulfate (MgSO₄) (99%) and sodium chloride (NaCl) (99%), were purchased from Merck (Darmstadt, Germany). Graphitized carbon black (GCB, 400 meshes) was obtained from Supelco. All glassware turned into rinsed with high purity acetone earlier than the use.
Sample Collection
On every sampling day, about 200 g grapes had been collected from each vine. The samples from three replicate treatments were pooled to make a sample size of 5 kg. The treated grapes have been analyzed at 0 (1 h), 1, 3, 5, 7, 10, 13, 16, 19 and 21 days of the application of the insecticide. Grapes were gathered at harvest time. Matured grapes had been analyzed at harvest. The accumulated grapes were placed in polyethylene bags and transferred to the laboratory after harvest and analyzed straight away.
Extraction procedure
The quick, easy, cheap, effective, rugged and safe (QuEChER) technique involves an acetonitrile partitioning and dispersive solid- phase extraction (d-SPE) which let in the simultaneous evaluation of a large range of insecticides in a variety of meals matrices. This technique was done as defined by other authors (5,19). According to this technique, the sample (1.00 kg of grapes) was chopped and homogenized for 5 min at high speed in a laboratory Homogenizer. A correctly weighed amount of 10.0 g of homogenized sample was placed into a 50 ml centrifuge tube with 10 ml of acetonitrile. The screw cap was closed and the tube was shaken vigorously for 1 min by hand, ensuring that the solvent interacted well with the entire sample. Then 4.00 g of anhydrous MgSO₄ and 1.00 g of NaCl was added, repeating the shaking process again for 1 min to prevent coagulation of MgSO₄. After centrifuging at 5000 × g for 5 min in 4◦C, the upper layer was cleaned by dispersive solid-phase extraction with 0.5 g of GCB and 1.50 g of anhydrous MgSO₄. The mixture was then shaken for 1 min and centrifuged for 5 min at 5000 ×g. The extract was filtered through a 0.45 µm PTFE filter and transferred to a vial. The cleaned extract sample was concentrated to 1.0 ml with a gentle stream of ultra-pure nitrogen gas and then 20 µL of this solution become injected into HPLC.
Apparatus
The residues of imidacloprid have been determined using HPLC (Shimadzu Corporation, Kyoto, Japan), version LC 10A Dual Pump with the UV-VIS detector using the reversed phase C-18, (Purospher Star RP-18, 250-4 mm i.d., 5 mm; Merck, Darmstadt, Germany) column. The mobile phase consisted of acetonitrile:water (40:60, v/v) with the solvent flow rate of 1mL min^-1 and the detector was set at a wavelength of 270 nm. The extent becomes 20 mL. The residues of imidacloprid were estimated in different substrates by comparison of the peak height of the sample with that of standard imidacloprid run under identical conditions. The percent recovery study of pesticide at different fortification levels was evaluated in order to assess the extraction efficiency of the method. Grape fruits from control plots had been spiked with imidacloprid at stages of 0.05, 0.10 and 0.20 mg kg^-1 in triplicate. The same extraction method and HPLC conditions were applied to both the sample analyses and recovery studies. The mean recoveries of imidacloprid fortified at these levels were found to be consistent and more than 89%, with relative standard deviation (RSD) values below 13%. The limit of detection (LOD) of 0.05 mg kg^-1 was obtained.
 

Results

The percent recovery of imidacloprid in grape berries is given in Table 1. By following the analytical method defined the recovery of imidacloprid residues in grape berries was in the range of 87-93.2%.

Table 1) Percent recovery of imidacloprid from spiked samples of grape

Substrate	Level of fortification (mg kg-1)	Recovery, % a (Mean±SD)
Grape samples	0.05	87 ± 4.32
	0.10	89 ± 3.58
	0.20	93 ± 5.04

^A Each value in the mean±standard deviation of the three replicates determinations

Following application of imidacloprid (Confidor 200SL) at 80 and 160 g ha^-1 1,000 L^-1 of water led to common initial deposits of 10.58 and 17.56 mg kg^-1 of imidacloprid on grape berries at single and double dosages, respectively. The results of imidacloprid residue analyses and the percent dissipation at different periodic intervals at single and double dosages are provided in Tables 2 and 3. These residue levels of imidacloprid dissipated to the extent of greater than 88% for the duration of one week.
Imidacloprid residue concentrations in grapes obtained in the dissipation observe with the corresponding first-order decay fits are supplied in Figs. 2 and 3. The half-life of imidacloprid in specific matrices was calculated the use of the first order rate equation: C_t=C₀e^_kt.
In which C_t represents the concentration of the pesticide residue at time t, C₀ represents the initial concentration and k is the rate constant per day. The half-life (t_1/2) was determined from the k value for each experiment, where t_1/2=ln2/k. The fitness of the data to first order kinetics was confirmed by testing the statistical significance of a correlation coefficient.
The degradation kinetics of this insecticide deposit were well described by first-order decay equation, (C(t)= 13.578× e^-0.333×t, R²=0.95) and (C(t)=73.558×e^-0.827×t, R²=0.95) for Imidacloprid at single and double dosages, respectively.
Figure 2) Residues of imidacloprid on grape following its application at 80 g a.i. ha^-1

Days after treatment	Residue level (mg kg_1)		Dissipation (%)
	Replication	Mean±SD
	ND*
Before applying	ND	ND	-
	ND
	11.24	10.58±2.42	-
0 (1 h after spray)	10.25
	10.26
	7.25	8.08±1.25	23.65
1	8.15
	8.85
	3.1	4.10±1.01	61.25
3	4.96
	4.25
	3.45	3.19±0.82	69.85
5	3.14
	2.98
	0.98	1.19±0.82	88.75
7	1.02
	1.58
	0.68	0.83±0.42	92.15
10	0.85
	0.96
	0.28	0.36±0.15	96.59
13	0.45
	0.35
	0.08	0.08±0.01	99.24
16	0.08
	ND
	ND	ND	-
19	ND
	ND	ND	-
	ND
21	ND
	ND

In keeping with our experimental results, the half-lives (T_1/2) of Imidacloprid are 2.21 and 2.94 days if applied on grape berries.
The theoretical dissipation models set up through regression between time after spray application and the corresponding residues in grape, correlation coefficient, and half-lives are provided in Table 4. The dissipation rate constants of 0.333 and 0.827 day^_1 corresponded to half-lives of 2.21 and 2.94 days, advised that the dissipation was dependent of the initial dose of imidacloprid and followed primary-order rate kinetics. Those consequences are consistent with the result of (11,25).

Figure 2) Dissipation of imidacloprid from grape following its application at 80 g a.i. ha^-1
 
 

Table 3) Residues of imidacloprid on grape following its application at 160 g a.i. ha^-1

Days after treatment	Residue level (mg kg_1)		Dissipation (%)
	Replication	Mean ± SD
	ND*
Before applying	ND	ND	-
	ND
	18.01	17.56±2.44	-
0 (1 h after spray)	17.95
	17.00
	10.84	10.65±2.01	39.35
1	10.54
	10.56
	5.89	6.22±1.54	64.57
3	5.94
	6.84
	3.98	3.42±1.87	80.52
5	3.28
	3.00
	1.95	2.35±1.08	86.61
7	2.62
	2.48
	0.91	0.94±0.04	94.64
10	1.01
	0.89
	0.41	0.37±0.06	97.89
13	0.38
	0.31
	0.05	0.05 ± 0.01	99.71
16	0.05
	0.05
	ND	ND	-
19	ND
	ND
	ND	ND	-
21	ND
	ND

 
 
 
 
Table 4) Theoretical dissipation models for Imidacloprid in grape after foliar treatment

Treatment (g ai kg_1)	Correlation Coefficient	Dissipation model	Half-life (Days)
	-0.9571	Y=13.578-0.333X	2.21
160	-0.958	Y=73.558-0.827X	2.94